Methods for the production of 3-o-deactivated-4&#39;-monophosphoryl lipid a (3d-mla)

ABSTRACT

Herein is disclosed a method for producing lipopolysaccharide (LPS), comprising: (a) growing a culture of deep rough mutant bacterial strain in a medium; (b) maintaining the culture in stationary phase for at least about 5 hr; (c) harvesting cells from the culture; and (d) extracting LPS from the cells. The method allows for the production of an LPS which can be used to produce a 3-O-deacylated monophosphoryl lipid A (3D-MLA) having at least about 20 mol % of the hexaacyl congener group. Also herein is disclosed a method of extracting lipopolysaccharide (LPS) from a culture of deep rough mutant bacterial strain cells, comprising: (a) extracting the cells with a solution consisting essentially of at least about 75 wt % of an aliphatic alcohol having from 1 to 4 carbon atoms and the balance water, thereby producing cells with reduced phospholipid content; and (b) extracting the cells with reduced phospholipid content with a solution comprising chloroform and methanol, thereby yielding a solution of LPS in chloroform and methanol (CM). This method provides LPS solutions in CM that have reduced phospholipid content and are produced by relatively simple and inexpensive process steps.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a non-provisional of and claims the benefit of U.S.Provisional Application No. 60/280,089, filed Mar. 30, 2001.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

NOT APPLICABLE

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of biosyntheticproduction of 3-O-deacylated-4′-monophosphoryl lipid A (3D-MLA). Moreparticularly, it concerns methods of improving the yield of desired3D-MLA congeners or minimizing the cost of purifying lipopolysaccharide(LPS) precursors of 3D-MLA.

2. Description of Related Art

It has long been recognized that enterobacterial lipopolysaccharides(LPS) are potent stimulators of the immune system. A variety ofresponses, both beneficial and harmful, can be elicited by submicrogramamounts of LPS. The fact that some of the responses are harmful, andsome of these can be fatal, has precluded clinical use of LPS per se. Ithas been observed that the component of LPS most responsible forendotoxic activity is lipid A.

Accordingly, much effort has been made towards attenuating the toxicattributes of LPS or lipid A without diminishing the immunostimulatorybenefits of these compounds. Notable among these efforts were those ofEdgar Ribi and his associates, which resulted in the production of thelipid A derivative 3-O-deacylated-4′-monophosphoryl lipid A (3D-MLA;compositions comprising 3D-MLA are commercially available under thetrade name MPL® from Corixa Corporation (Seattle, Wash.)), 3D-MLA hasbeen shown to have essentially the same immunostimulatory properties aslipid A but lower endotoxicity (Myers et al., U.S. Pat. No. 4,912,094).Myers et al. also reported a method for production of 3D-MLA, asfollows. First LPS or lipid A obtained from a deep rough mutant strainof a gram-negative bacterium (e.g. Salmonella minnesota R595) isrefluxed in mineral acid solutions of moderate strength (e.g. 0.1 N HCl)for a period of approximately 30 min. This leads to dephosphorylation atposition 1 of the reducing-end glucosamine and decarbohydration at the6′ position of the non-reducing glucosamine of lipid A. Second, thedephosphorylated decarbohydrated lipid A (a.k.a. monophosphoryl lipid Aor MLA) is subject to base hydrolysis by, for example, dissolving in anorganic solvent such as chloroform:methanol (CM) 2:1 (v/v), saturatingthe solution an aqueous solution of 0.5 M Na₂CO₃ at pH 10.5, and flashevaporating solvent. This leads to selective removal of theβ-hydroxymyristic acid moiety at position 3 of the lipid A, resulting in3-O-deacylated -4′-monophosphoryl lipid A (3D-MLA).

The quality of the 3D-MLA produced by the above method is highlydependent on the purity and composition of the LPS obtained from thegram-negative bacterium. For one example, the lipid A component of LPSis a mixture of closely related species that contain between about 5-7fatty acid moieties. In the formation of 3D-MLA, as is clear from theabove discussion, one fatty acid moiety is removed, yielding 3D-MLA withbetween about 4-6 fatty acid moieties. It is generally held that 3D-MLAwith at least 6 fatty acid moieties is preferred in terms of thecombination of maintained or enhanced immunostimulatory benefits,reduced toxicity, and other desirable properties (Qureshi and Takayama,in “The Bacteria,” Vol. XI (Iglewski and Clark, eds.), Academic Press,1990, pp. 319-338).

For another example, commercial scale extraction of LPS fromgram-negative bacteria typically involves the Chen method (Chen et al.,J. Infect. Dis. 128:543 (1973)); namely, extraction with CM, which leadsto an LPS- and phospholipid-rich CM phase from which LPS can later bepurified. However, purification of LPS from the LPS- andphospholipid-rich CM phase typically requires multiple precipitationsteps to obtain LPS of sufficient purity for use in immunostimulatoryapplications such as, for example, use as a vaccine adjuvant.

Therefore, it would be desirable to have methods for convenientlypreparing highly pure LPS compositions. Further, it would be desirableto have methods for generating LPS compositions which compositions have3D-MLA with increased levels of hexaacyl congeners.

Known fermentation techniques have been used to prepare cultures ofgram-negative bacteria comprising readily purifiable LPS. These knowntechniques typically involve harvesting of bacterial cultures at earlystationary phase, in keeping with standard bacteriological practices.However, it has been observed that the degree of acylation of LPSproduced according to known conditions is variable. For example, thecontent of heptaacyl species in the lipid A of S. minnesota R595 canvary from 20% to 80%, depending on the batch (Rietschel et al., Rev.Infect. Dis. 9:S527 (1987)). This variability in heptaacyl congenercontent would result in the significant differences in the hexaacylcongener content in the 3D-MLA prepared from these LPS batches.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a method forproducing lipopolysaccharide (LPS), comprising:

-   -   (a) growing a culture of a deep rough mutant bacterial strain in        a medium;    -   (b) maintaining the culture in stationary phase for at least        about 2 hr;    -   (c) harvesting cells from the culture; and    -   (d) extracting LPS from the cells.

The method allows for the production of an LPS that yields 3D-MLA with arelatively high proportion (i.e. at least about 20 mol %) of congenerscomprising 6 fatty acid moieties.

In another embodiment, the present invention relates to a method ofextracting lipopolysaccharide (LPS) from a culture of deep rough mutantbacterial strain cells, comprising:

(a) extracting the cells with a solution consisting essentially of atleast about 75 wt % of an aliphatic alcohol having from 1 to 4 carbonatoms and the balance water, thereby producing cells with reducedphospholipid content;

(b) extracting the cells with reduced phospholipid content with asolution comprising chloroform and methanol (CM), thereby yielding asolution of LPS in CM.

This method provides LPS solutions in CM that have reduced phospholipidcontent and that are therefore well-suited to further modification andpurification to 3D-MLA. The method involves relatively simple andinexpensive steps.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows TLC plates of ethanol extracts and LPS samples obtainedwith different temperatures during the ethanol extractions. The plate onthe left shows, going from left to right, the ethanol extracts attemperatures of 22° C., 37° C., and 50° C. The sample at the far rightof this plate is an authentic LPS sample. The plate on the right showsthe LPS obtained from each preparation. The samples in lanes 3, 4, and 5correspond to LPS from cells subjected to pre-extractions with ethanolat 22° C., 37° C., and 50° C., respectively. The heavy bands atR_(f)˜0.6 correspond to phospholipid and fatty acid impurities. Thelevels of these impurities are reduced by increasing the temperature ofthe ethanol extractions, and are very low in the sample that waspre-extracted at 50° C.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one embodiment, the present invention relates to a method forproducing lipopolysaccharide (LPS), comprising:

(a) growing a culture of a deep rough mutant bacterial strain in amedium;

(b) maintaining the culture in stationary phase for at least about 2 hr;

(c) harvesting cells from the culture; and

(d) extracting LPS from the cells.

Lipopolysaccharides are the main lipid constituent in the outer leafletof the outer membrane of gram-negative bacteria. The lipopolysaccharidefraction of a gram-negative bacterium comprises, among other components,lipid A. As has been described, lipid A can be decarbohydrated andpartially dephosphorylated to yield monophosphoryl lipid A (MLA), andMLA can be selectively deacylated at position 3 to yield3-O-deacylated-4′-monophosphoryl lipid A (3D-MLA).

However, lipid A produced by gram-negative bacteria typically comprisesa number of species that have the same overall lipid A structure butdiffer in the number of fatty acid moieties they contain. Groups oflipid A species with the same number of fatty acids are referred toherein as “congeners.” Lipid A congeners having from 4 to 7 fatty acidmoieties are produced by standard commercial-scale culturing ofgram-negative bacteria such as S. minnesota R595. As a result, the3D-MLA produced from, e.g., S. minnesota R595 lipid A has a congenercomposition typically ranging from 3 to 6 fatty acid moieties (because3D-MLA has undergone loss of one fatty acid moiety).

Heterogeneity in 3D-MLA (via lipid A and MLA) congener composition isattributable to two sources: (1) biosynthetic variability in theassembly of the lipid A and (2) loss of fatty acid moieties from thelipid A backbone during processing to 3D-MLA. Though not to be bound bytheory, biosynthetic variability is believed to occur because ofnon-absolute substrate specificity of the acyltransferases involved inthe terminal steps of lipid A biosynthesis, among other explanations.Loss of fatty acid moieties from the lipid A backbone may also occurduring the acid and alkaline hydrolyses typically used in 3D-MLAproduction.

Surprisingly, it was discovered that 3D-MLA congener composition can bealtered by altering the parameters of a process of culturing a deeprough mutant bacterial strain that produces lipid A. Specifically, itwas discovered that maintaining the culture of the deep rough mutantbacterial strain at stationary phase for at least about 5 hr prior toharvesting results in a change in the proportions of lipid A congenersso produced such that, typically, at least about 20 mol % of the 3D-MLAlater produced from the lipid A contains 6 fatty acids. Preferably, atleast about 50 mol % of the 3D-MLA contains 6 fatty acids. A maintenanceat stationary phase time of about 5.5 hr has been found to beparticularly effective. This is in distinction to the typical culturingprocesses known in the art, wherein harvesting occurs almost immediatelyafter entry of the culture into the stationary phase; in the knownprocess, the congener content of the LPS is highly variable and resultsin 3D-MLA with variable hexaacyl congener content.

By “deep rough mutant bacterial strain” is meant a strain of agram-negative bacterium having a deep rough phenotype. A “deep rough”phenotype means that the polysaccharide moiety attached to the lipid Aconsists of only about 2-3 residues of 2-keto-3-deoxy-D-mannooctulonicacid (KDO). Preferably, the deep rough mutant bacterial strain isselected from the genus Salmonella. More preferably, if the deep roughmutant bacterial strain is of genus Salmonella, it is of speciesSalmonella minnesota, and even more preferably, it is strain Salmonellaminnesota R595. Other deep rough mutant bacterial strains, such asProteus mirabilis strains, among others, can be used.

Any technique appropriate for growing a deep rough mutant bacterialstrain can be used. Typically, this will involve the use of at least onecommercial-scale bioreactor. In one embodiment, the technique involvesinoculating a relatively small (e.g. 15 L) bioreactor with cells of thedeep rough mutant bacterial strain, growing the deep rough mutantbacterial strain until a stationary phase, followed by aseptic transferof the 15-L cell broth to a large (e.g. 750 L) bioreactor.

The growing can be performed on any medium known or discovered to allowthe growth of the deep rough mutant bacterial strain. In one preferredembodiment, the medium is M9, a mixture of inorganic salts supplementedwith dextrose and casamino acids. The composition of M9 is well-known toone of ordinary skill in the art.

After the deep rough mutant bacterial strain has been maintained atstationary phase for at least about 5 hr, the cells can be harvestedfrom the culture and LPS extracted from the cells. Known techniques maybe employed to harvest cells from the culture and extract LPS from thecells, although a preferred technique for extracting LPS from the cellsis described below.

Harvesting can be performed by any known technique. In one preferredembodiment, after the cell culture has been maintained at stationaryphase for at least about 5 hr, the contents of the bioreactor are pumpedto a tangential filtration apparatus to separate spent medium from thecells.

The LPS is then extracted from the cells by any appropriate technique.Known techniques include the Galanos method, which involves extractingLPS with a mixture of phenol, chloroform, and petroleum ether (PCP),followed by evaporation of the chloroform and petroleum ether, additionof acetone and water to precipitate LPS, and recovery of LPS bycentrifugation or filtration (Galanos et al., Eur. J. Biochem. 9:245(1969)), and the Chen method, cited above, which involves extracting LPSwith a mixture of chloroform and methanol (CM), followed by a series ofmethanol precipitation steps.

An improvement of the Chen method is described below, and is preferredfor manufacture of LPS and its derivatives for commercial applications.

Regardless of the extraction technique, the result is a substantiallypure dried LPS, which can be further processed by sequential acidhydrolysis and base hydrolysis to form 3D-MLA, as is taught by Ribi,U.S. Pat. No. 4,436,727, and Myers et al., U.S. Pat. No. 4,912,094,which are hereby incorporated herein by reference. To summarize theteachings of these references as a preferred embodiment for theformation of 3D-MLA, the LPS is reacted with an organic or inorganicacid, and then lyophilized to produce MLA. The inorganic acid ispreferably hydrochloric acid, sulfuric acid, or phosphoric acid. Theorganic acid is preferably toluene sulfonic acid or trichloroaceticacid. The reaction may be performed at a temperature between about 90°C. and about 130° C. for a sufficient time to complete hydrolysis,commonly between about 15 min and about 60 min. The MLA may be treatedwith a solvent, preferably acetone, to dissolve fatty acids and otherimpurities, and the impurity-rich fatty acid solvent is removed.

Thereafter, the MLA is subjected to mild alkaline treatment toselectively remove the β-hydroxymyristic acid from position 3 of the MLA(under mild alkaline conditions, only the β-hydroxymyristic acid atposition 3 in labile). The mild alkaline treatment can be carried out inaqueous or organic media. Appropriate organic solvents include methanolor other alcohols, dimethyl sulfoxide (DMSO), dimethylformamide (DMF),chloroform, dichloromethane, or mixtures thereof, among others.Combinations of water and organic solvents miscible with water may alsobe employed.

The Alkaline base used to perform the hydroloysis is preferably selectedfrom hydroxides, carbonates, phosphates, or amines. Illustrativeinorganic bases include sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium bicarbonate, sodium bicarbonate, and potassiumbicarbonate, among others. Illustrative organic bases include alkylamines (such as diethylamine and triethylamine, among others), amongothers.

In aqueous media, the pH is typically between about 10 and about 14 ,preferably between about 10 and about 12. The hydrolysis reaction istypically performed at from about 20° C. to about 80° C., preferablyfrom about 50° C. to about 60° C., for a period of about 10 min to about48 hr.

One preferred technique for alkaline hydrolysis involves dissolving MLAin CM 2:1 (v/v), saturating the solution with an aqueous buffer of 0.5 MNa₂CO₃ at pH 10.5, and then flash evaporating the solvent at 45-50° C.under a vacuum aspirator (approximately 100 mm Hg).

In another embodiment, the present invention relates to a method ofextracting lipopolysaccharide (LPS) from a culture of deep rough mutantbacterial strain cells, comprising:

(a) extracting the cells with a solution consisting essentially of atleast about 75 wt % of an aliphatic alcohol having from 1 to 4 carbonatoms and the balance water, thereby producing cells with reducedphospholipid content;

(b) extracting the cells with reduced phospholipid content with asolution comprising chloroform and methanol, thereby yielding a solutionof LPS in chloroform and methanol.

The deep rough mutant bacterial strain cells, the culture thereof, andmethods of preparing the culture are as described above. Preferably, thedeep rough mutant bacterial strain is selected from the generaSalmonella or Escherichia. More preferably, if the deep rough mutantbacterial strain is of genus Salmonella, it is of species Salmonellaminnesota, and even more preferably, it is strain Salmonella minnesotaR595. If the deep rough mutant bacterial strain is of genus Escherichia,more preferably it is of species Escherichia coli, and more preferablyit is strain Escherichia coli D31m4.

The first extracting step can be performed with any short chainaliphatic alcohol. The aliphatic alcohol can be linear, branched, orcyclic. Preferably, the aliphatic alcohol has from 2 to 4 carbon atomsand is miscible with water. More preferably, the aliphatic alcohol isethanol.

The solution comprising the aliphatic alcohol can comprise anyproportion of aliphatic alcohol of 75 wt % or greater. Preferably, thesolution comprises between about 85 wt % and about 95 wt % aliphaticalcohol. Essentially, the balance of the solution is water. Traces ofother compounds may be present as a result of incomplete purification orother contamination of the aliphatic alcohol and water components of thesolution.

The temperature at which the first extracting step is performed can beany temperature which is effective in providing sufficient extraction ofphospholipid from the cultured cells. Preferably, the temperature isbetween about 35° C. and about 65° C. More preferably, the temperatureis between about 45° C. and about 55° C.

Other parameters of the first extracting step, such as rate of additionof the aliphatic alcohol solution, duration of contact of the solutionand the cells, and agitation or lack thereof, among others, can beroutinely varied by one of ordinary skill in the art.

The first extracting step results in (i) a phospholipid-rich aliphaticalcohol solution phase and (ii) cells with a reduced phospholipidcontent. The LPS component of the cell membranes segregatessubstantially completely with the cells with a reduced phospholipidcontent.

The second extracting step involves extracting the cells with a reducedphospholipid content with a solution of chloroform:methanol (CM).

Any proportion of chloroform and methanol known to be suitable for usein extracting LPS from cell membranes (such as in the Chen method) maybe used in the second extracting step. Typically, the proportion ofchloroform to methanol is from about 2:1 to about 9:1. Solvent mixtureswith properties equivalent to those of CM may also be used to obtain LPSfrom the cells with a reduced phospholipid content.

An advantage of the present method over the Chen method lies in theremoval of phospholipid in the first extracting step. Whereas the CMextraction of the Chen method results in an LPS solution that containssubstantial levels of phospholipids, the second extracting step of thepresent invention, being performed on cells with a reduced phospholipidcontent, results in an LPS-rich solution that is substantially devoid ofphospholipid. Alternative methods of producing LPS preparations that arerelatively free of phospholipids, such as the method of Galanos (seeabove), are less desirable because they are not amenable to large scaleproduction, they use solvent mixtures that pose health and safetyconcerns (e.g. phenol:chloroform:petroleum ether), or both.

Given the substantial absence of phospholipid from the LPS solution,further purification of the LPS according to this method is generallysimpler and less expensive than under the Chen method. It has been foundthat a dry LPS residue of sufficient purity can be formed by evaporatingthe chloroform and methanol from the LPS solution.

Optionally, the LPS can be further processed, such as by the acidhydrolysis and base hydrolysis steps described above, to produce MLA or3D-MLA.

The 3D-MLA produced by following the methods described above can be usedfor a variety of purposes. One preferred use is as an immunostimulant oradjuvant for pharmaceutical compositions comprising an immunogenicpolynucleotide, polypeptide, antibody, T-cell, or antigen-presentingcell (APC). An immunostimulant or adjuvant refers to essentially anysubstance that enhances or potentiates an immune response (antibodyand/or cell-mediated) to an exogenous antigen.

One immune response which the MLA or 3D-MLA produced according to thepresent invention may stimulate is the Th1 type. A combination ofmonophosphoryl lipid A (MLA), preferably 3-de-O-acylated monophosphoryllipid A (3D-MLA), together with an aluminum salt has been observed to beeffective as an adjuvant for eliciting a predominantly Th1-typeresponse. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell-mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a pharmaceuticalcomposition comprising MLA or 3-D-MLA, a patient will support an immuneresponse that includes Th1- and Th2-type responses. When the response ispredominantly Th1-type, the level of Th1-type cytokines will increase toa greater extend than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A (MLA), preferably 3D-MLA, with asaponin derivative (such as Quil A or derivatives thereof, includingQS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); Escin;Digitonin; or Gypsophila or Chenopodium quinoa saponins), such as thecombination of QS21 and 3D-MLA adjuvant, as described in WO 94/00153, ora less reactogenic composition where the QS21 is quenched withcholesterol, as described in WO 96/33739. One preferred formulationscomprise an oil-in-water emulsion and tocopherol. Another particularlypreferred adjuvant formulation employing QS21, 3D-MLA, and tocopherol inan oil-in-water emulsion is described in WO 95/17210.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 General Methods

A. Media Preparation

Cell growth was conducted in M9 medium, which is prepared by combiningsterile solutions of inorganic salts, casamino acids, and dextrose. TheM9 salt solution is typically prepared in the fermentor and contains thefollowing salts: 2.0 g/L NaCl, 0.2 g/L MgSO₄.7H₂O, 3.0 g/L KH₂PO₄, 6.0g/L Na₂HPO₄, and 1.0 g/L NH₄Cl. Sterile solutions of 20% (w/v) casaminoacids (20 mL/L) and 50% (w/v) dextrose (32 mL/L) and then addedaseptically to the fermentor to yield the completed medium.

B. Seed Growth

Typically, a sterile 250 mL Erlenmeyer flask was charged with 50 mLsterile M9 medium. A seed vial of Salmonella minnesota R595 (ca. 10⁸cfu) was thawed and added to the flask, which was then stoppered with agauze plug. The culture was incubated at 37° C. for 6-8 h, until robustgrowth is evident.

C. Cell Growth

Cultures of Salmonella minnesota R595 were grown in a BioFlo IIIfermentor (New Brunswick Scientific, Inc.) equipped with a 2.5 L glassvessel. In a typical run, the vessel was charged with 2.0 L of M9 saltssolution, autoclaved, and sterile solutions of casamino acids anddextrose were then added aseptically. The fermentor was equipped withfeedlines for antifoam (0.1% SAG-471, Witco Corp.) and NH₄OH (30%) aswell as probes for pH, dO₂, and foam. The medium was adjusted to pH 6.9using the NH₄OH feed. The fermentor was then inoculated with the entireseed culture and was incubated at 37° C. with air sparging (typically2.0 Lpm) and stirring (typically 50 rpm). The growth phase of theculture was monitored by measuring optical density at 590 nm. Cells wereharvested by either centrifugation or tangential flow filtration, washedwith water, and lyophilized.

D. Extraction of Lipopolysaccharide (LPS)

LPS was isolated according to the procedure of Qureshi et al. (1986)with minor modifications. In a typical run, the dried cells were firststirred at a concentration of 20 mg/mL in 90% ethanol (v/v) at roomtemperature for 1 h and were then recovered by vacuum filtration. Thecells were subjected to a second ethanol extraction followed bysequential extractions with acetone and diethyl ether (15 min each, bothat 40 mg/mL based on initial weight), and the resulting ether powder wasallowed to air dry overnight. Meanwhile, a solution of phenol(89%):chloroform:petroleum ether 19:45:72 (v/v/v; abbreviated PCP) wasprepared and allowed to stand overnight. The ether powder was suspendedin PCP, which was decanted off of the excess water, at a concentrationof 70 mg/mL. The solution was stirred for 30 min and then wascentrifuged (3000×g, 15 min, 0-5° C.). The supernatant fraction wasdecanted into a round bottom flask and the cell pellet was extracted asecond time with PCP. The supernatant fractions were combined and rotaryevaporated at 40° C. until all volatile solvents were largely removed.The remaining volume was then measured. Water was added dropwise until apersistent turbidity was evident, and then 5 volumes acetone followed by1 volume diethyl ether (both chilled in an ice bath) were added to thephenol solution with rapid mixing. The solution was placed in an icebath for 30 min and then the precipitated LPS was recovered bycentrifugation (5000×g, 15 min, 0-5° C.). It was generally necessary togravity filter the supernatant fraction to recover any LPS that did notremain in the pellet. The LPS was washed one time in a minimal volume ofcold acetone, recovered by centrifugation/filtration, and was then driedunder vacuum. Typical yields were 4-5% based on the initial dry weightof cells.

E. Preparation of 4′-monophosphoryl Lipid A (MLA)

LPS was suspended in water at a concentration of 10 mg/mL, using bathsonication at 45-55° C. to aid in dispersing the solid material. Theresulting solution should be slightly turbid with no solid visible tothe unaided eye. To this solution was added 1 volume of 0.2 N HCl, andit was then placed in a boiling water bath for 15 min. The reaction wasquenched in an ice bath, and then was extracted with 5 volumes (relativeto the initial LPS solution) of chloroform:methanol 2:1 (v/v). Thebiphasic solution was vortexed and the phases were separated by lowspeed centrifugation (500-1000×g). The lower phase was recovered andevaporated under nitrogen, yielding crude MLA.

F. Preparation of 3-O-deacylated-4′-monophosphoryl Lipid A (3D-MLA)

Crude MLA was dissolved in chloroform:methanol 2:1 (v/v) at aconcentration of between about 1-5 mg/mL, and 3.0 mL of this solutionwas transferred to a 16×100 mm test tube. An additional 0.4 mL ofmethanol was added to the tube, and it was then placed in a water bathat 50° C. for 10 min. The reaction was initiated by addition of 40 μL0.5 M KHCO₃, pH 10.5, and the solution was incubated at 50° C. for 20min. At the end of this time, the tube was removed from the water bathand the reaction was quenched by addition of 2.0 mL 0.1 N HCl (chilled)followed by vortexing. 3D-MLA was recovered by addition of 1.0 mLmethanol, vortexing, centrifugation (500-1000×g), and evaporation of thelower (organic) phase to dryness under nitrogen.

EXAMPLE 2 Analytical Methods

A. Thin Layer Chromatography (TLC) of MLA and Related Samples

All TLC analyses were carried out using 5×10 cm plates coated withSilica Gel 60 (E Merck). Samples were generally applied to the TLCplates as 10 mg/mL solutions in chloroform:methanol 4:1 (v/v), with 3 μLsolution (30 μg sample) applied in small spots to a 5 mm line using acapillary pipette. Plates were developed with a solvent systemcomprising chloroform/methanol/water/ammonium hydroxide 50:31:6:2 (v/v).Bands on the developed plates were visualized by spraying with asolution of 10% (w/v) phosphomolybdic acid in ethanol followed bycharring at 150-160° C. In some cases, relative intensities of spotswere quantified by scanning densitometry with a Shimadzu CS9000U DualWavelength Flying Spot Scanner (Shimadzu Corp.), using a scanningwavelength of 520 nm.

B. Analysis of MLA/3D-MLA by High Performance Liquid Chromatography(HPLC)

Samples to be analyzed were first converted to the free acid form bywashing a solution of 3-5 mg sample in 5 ml chloroform:methanol (2:1v:v) with 2 ml 0.1 N HCl. The biphasic system was vortexed, centrifuged,and the lower (organic) phase was transferred to a test tube andevaporated under a stream of nitrogen. The residue was then methylatedby treatment with diazomethane. Briefly, an ethereal solution ofdiazomethane was prepared by placing 60-100 mg1-methyl-3-nitro-1-nitrososguanidine (MNNG; Aldrich) in a 2 dram vial,adding 60 μL diethyl ether per mg MNNG, then adding 9 μL 5 N NaOH per mgMNNG while stirring the solution at <−10° C. Following completion of thereaction, the lemon yellow ether phase was dried by transferring it to asecond vial that contained several pellets of NaOH and swirling, allwhile at <−10° C. The acid-washed sample was dissolved in 1 mlchloroform:methanol 4:1 (v:v), placed in a bath at <−10° C., anddiazomethane solution was added dropwise with stirring until a faintyellow tint persists. The solvent was then evaporated at ambienttemperature under a stream of nitrogen and was further dried undervacuum for at least 30 min.

Chromatographic analyses were conducted on a C₁₈ reverse phase column(Nova-Pak, 4 μm particle size, 8 mm×10 cm [Waters]). Methylated sampleswere dissolved in chloroform:methanol 4:1 (v/v) at a concentration of100 μg/mL and passed through a 0.45 μm PTFE syringe filter. An injectionvolume of 20-25 μL was typically used, followed by elution with a lineargradient of 20 to 80% isopropanol in acetonitrile over 60 min at a flowrate of 2 ml/min with monitoring at 210 nm.

C. Analysis of LPS Congener Content by HPLC

LPS tends to be a highly heterogeneous material due to variability in 1)the number of sugar residues in the O-antigen and core regions, 2) polarsubstitutions in the core region and on the phosphates in the lipid A,and 3) the number and location of fatty acids attached to the lipid Abackbone. It is this latter source of variability that is of interestrelative to the congener content of 3D-MLA (MPL®). Hydrolysis of LPS toMLA and 3D-MLA removes variability in the O-antigen and core regions,however it also introduces additional heterogeneity due to controlledloss of O-linked fatty acids. This prevents the acylation pattern in theintact LPS from being accurately known. As a way around this, a methodwas developed wherein the phosphates and the core region are removedunder mild conditions that do not result in loss of O-linked fattyacids. The resulting dephosphorylated lipid A (zero phosphoryl lipid A,or ZPL) can then be analyzed by HPLC, yielding an accurate reflection ofthe acylation pattern in the parent LPS.

The method was typically carried out as follows. Between 0.5-5.0 mg ofLPS sample was hydrolyzed in 200 μL concentrated hydrofluoric acid for3-4 h at 27° C. This reaction must be done in a tightly capped Teflontube and in a well-ventilated fume hood. The HF was removed byevaporation under a stream of nitrogen at ambient temperature, and thehydrolysate was then dissolved in chloroform:methanol 4:1 (v/v) andtransferred to a 16×100 mm glass test tube, and solvent was evaporatedunder a stream of nitrogen. The residue was suspended in 1.0 mL 0.1%triethylamine using bath sonication, 1.0 mL 40 mM NaOAc was added, andthe tube was suspended in a boiling water bath for 30-45 min. Thereaction was quenched by cooling in an ice bath and the ZPL wasrecovered by extraction with 5 mL chloroform:methanol 2:1 (v/v). Theorganic phase was transferred to a small screw cap vial and solvent wasevaporated under nitrogen. The ZPL was derivatized by adding 200 μL of10 mg/mL O-(3,5-dinitrobenzyl)hydroxylamine HCl (Regis Technologies,Inc.) in pyridine, tightly capping the vial, then incubating at 60° C.for 3 h. Pyridine was evaporated under nitrogen and the residue wasfurther dried under vacuum for >30 min. The residue was then suspendedin 500 μL chloroform:methanol 2:1 (v/v) and loaded onto a 0.5-1.0 mL bedof Accell-QMA (acetate form; Waters) that had been pre-equilibrated inthe same solvent. The column was rinsed with a total of 5.0 mLchloroform:methanol 2:1 (v/v) in several small portions and the eluatewas collected in a 16×100 mm test tube. 2.0 mL 0.1 N HCl was added tothe eluate, the biphasic system was vortexed, centrifuged briefly at500-1000×g, and the lower (organic) phase was transferred to a anothertest tube and evaporated under nitrogen. The residue was dissolved in100-300 μL chloroform:methanol 4:1 (v/v) and filtered through a 0.45 μMPTFE syringe filter. The filter was rinsed twice withchloroform:methanol 4:1 (v/v) and the filtrate was evaporated undernitrogen. The filtrate was finally taken up in 50-150 μLchloroform:methanol 4:1 (v/v) and transferred to an autoinjector vialfor HPLC analysis. HPLC conditions were as follows: C₁₈ reverse phasecolumn (e.g., Waters), 10 μL injection volume, linear gradient 20 to 80%isopropanol in acetonitrile over 60 min at a flow rate of 2 ml/min,monitor at 254 nm.

EXAMPLE 3 Comparison of Congener Composition of MLA/3D-MLA from CulturesHarvested at Different Times

A series of fermentor runs was conducted with the following parameters:2.0 L M9 medium (initial pH 6.84-6.87), 2 Lpm air flow, stirring at 50rpm, 37° C., no pH control. Cultures were monitored by measuring opticaldensity at 590 nm and were stopped when the desired growth stage wasattained. Cells were processed and extracted as described above to yieldLPS samples, which were then hydrolyzed to MLA and 3D-MLA and analyzedby HPLC (see Examples 1 and 2). Results are summarized in Table 1. TABLE1 Congener composition of MLA and 3D-MLA from cells harvested atdifferent ages. MLA 3D-MLA 3-O- 3-O- Culture age Time in deacylateddeacylated Run Description at harvest stationary phase hexaacylheptaacyl hexaacyl A Late exponential 6.75 h  N/A 12.4% 12.2% 9.9% BEarly stationary 9.5 h ˜0.5 h 9.2% 6.8% 9.2% phase C Late stationary  15h   ˜6 h 19.5% 13.2% 21.5% phase

The data show that cultures of S. minnesota R595 alter the acylationpattern of their LPS during stationary phase, resulting in an increasein the overall content of 3-O-deacylated hexaacyl plus heptaacyl speciesin MLA derived from this LPS, and this in turn gives rise to increasedcontent of 3-O-deacylated hexaacyl species in 3D-MLA prepared from thisMLA.

EXAMPLE 4 Comparison of Congener Composition of LPS from CulturesHarvested at Different Times

A series of fermentor runs was conducted with the following parameters:2.0 L M9 medium (initial pH 6.84-6.87), 2 Lpm air flow, stirring at 225rpm, 37° C., no pH control. The growth stage of the cultures wasmonitored by measuring optical density at 590 nm. Cells were processedand extracted as described in Example 1 to yield LPS samples. LPSsamples were hydrolyzed to ZPL and analyzed by HPLC as described inExample 2. Results are summarized in Table 2. TABLE 2 Congenercomposition of LPS from cells harvested at different ages. Congenercontent Culture Time in 3-O- age at time stationary 3-O-acyl deacylatedRun Description of harvest phase hexaacyl hexaacyl heptaacyl A Early  9h ˜0.5 h 76% 0% 24% stationary phase B Late 15 h   ˜6 h 48% 17%  19%stationary phase

No 3-O-deacylated hexaacyl component was detected in the LPS from theearly stationary phase cells (run A). Thus, the only source ofhexaacylated congeners in 3D-MLA prepared from this LPS would be theheptaacylated material (24%). In contrast, LPS from cells harvested atlate stationary phase contained both heptaacyl and 3-O-deacylatedhexaacyl species (19% and 17%, respectively). Both of these specieswould contribute to the hexaacyl content in 3D-MLA (MPL®) prepared fromthis LPS. It was unexpected to find that cells produce 3-O-deacylatedhexaacyl LPS species under certain conditions.

EXAMPLE 5 Effect of Pre-extraction Temperature on Purity of S. minnesotaR595 LPS

Cells of S. minnesota R595 were grown in an 80 L fermentor (NewBrunswick Scientific) using essentially the same conditions as outlinedin Example 1. The cells were concentrated by tangential flow filtrationbut were not centrifuged, and the slurry contained 51.5 mg dry cell massper mL. Three solutions were prepared in which 150 mL aliquots of thecell suspension were each combined with 600 mL ethanol. The ethanolsolutions were stirred for 1 h at 22° C., 37° C., and 50° C. and werefiltered. The cells were subjected to a second ethanol extraction underthe same conditions except using 95% ethanol. The cells were recoveredby suction filtration and were then extracted overnight inchloroform:methanol 4:1 (v/v) at 50° C. The solutions were filtered andthe filtrates were rotary evaporated to dryness, yielding the LPSpreparations. Samples of the first and second ethanol extractionfiltrates obtained at each temperature as well as the LPS obtained fromthe pre-extracted cells were analyzed by thin layer chromatographyaccording to the method in Example 2. Images of the TLC plates are shownin FIG. 1.

FIG. 1 shows TLC plates of ethanol extracts and LPS samples obtainedwith different temperatures during the ethanol extractions. The plate onthe left shows, going from left to right, the ethanol extracts attemperatures of 22° C., 37° C., and 50° C. The sample at the far rightof this plate is an authentic LPS sample. The plate on the right showsthe LPS obtained from each preparation. The samples in lanes 3, 4, and 5correspond to LPS from cells subjected to pre-extractions with ethanolat 22° C., 37° C., and 50° C., respectively. The heavy bands atR_(f)˜0.6 correspond to phospholipid and fatty acid impurities. Thelevels of these impurities are reduced by increasing the temperature ofthe ethanol extractions, and are very low in the sample that waspre-extracted at 50° C.

It is apparent from the TLC plates in FIG. 1 that pre-extraction withethanol at elevated temperatures is effective at removing impuritiesthat are otherwise co-extracted with chloroform:methanol 4:1 (v/v).Pre-extraction at 50° C. results in LPS that is largely free of theseimpurities.

EXAMPLE 6 Comparison of LPS Obtained by with and without Pre-extractionwith Ethanol

Three batches of cells of S. minnesota R595 were grown in a 750 Lfermentor (B. Braun) using essentially the same conditions as outlinedin Example 1. Cells were harvested by tangential flow filtration, and asample of the cell suspension was obtained from each batch andlyophilized. The bulk of the cells were subjected to two pre-extractionswith 90% ethanol at 50° C. for 1 hr. Cells were recovered by tangentialflow filtration between extractions. The cells were then extractedovernight with chloroform:methanol 4:1 (v/v) at reflux. The extract wasrecovered by tangential flow filtration and evaporated to dryness. Thelyophilized cell samples were extracted overnight in refluxingchloroform:methanol 4:1 (v/v), and the solutions were filtered and thefiltrates were evaporated to dryness. LPS samples obtained with andwithout ethanol pre-extraction was analyzed by TLC essentially asdescribed in Example 2. TLC plates were scanned from about R_(f)=0.01 to0.90, and the ratio of intensity in the LPS region (R_(f)=0.01 to 0.020)to total intensity was calculated for each sample. The results are givenin Table 3. TABLE 3 LPS purity from cells with and withoutpre-extraction with ethanol. Percent of total intensity in LPS region¹without ethanol with ethanol Run Lot Number pre-extractionpre-extraction A 48020-B2698C 7 86 B 48020-C0598A 11 83 C 48020-C0598B14 88Note:¹Percent of total intensity in LPS region = [(intensity in R_(f) = 0.01to 0.20)/(intensity in R_(f) = 0.01 to 0.90)] × 100

The results in Table 3 demonstrate that the LPS obtained followingpre-extraction of S. minnesota R595 cells with 90% ethanol at 50° C. issubstantially purer than material from cells without pre-extraction.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the steps or in the sequenceof steps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1-25. (canceled)
 26. A composition of 3D-MLA comprising at least about20 mol % of hexaacyl congener.
 27. A composition according to claim 26comprising between 20 and 50 mol % of hexaacyl congener
 28. Acomposition according to claim 26 comprising at least 30 mol % ofhexaacyl congener.
 29. A composition according to claim 26 comprising21.5% hexaacyl congener.
 30. A composition of LPS comprising at leastabout 20 mol % of a combination of heptaacyl congener and 3-O-deacylatedhexaacyl congener.
 31. A composition according to claim 30 comprising atleast 30 mol % of a combination of heptaacyl congener and 3-O-deacylatedhexaacyl congener.
 32. A composition according to claim 30 comprising36% of a combination of heptaacyl congener and 3-O-deacylated hexaacylcongener.
 33. A composition of MLA comprising at least about 20 mol % ofa combination of heptaacyl congener and 3-O-deacylated hexaacylcongener.
 34. A composition according to claim 33 comprising at least 30mol % of a combination of heptaacyl congener and 3-O-deacylated hexaacylcongener.
 35. A composition according to claim 33 comprising 32.7% of acombination of heptaacyl congener and 3-O-deacylated hexaacyl congener.36. A composition according to claim 26 wherein said 3D-MLA is extractedfrom a deep rough mutant strain of a gram-negative bacterium.
 37. Acomposition according to claim 30 wherein said LPS is extracted from adeep rough mutant strain of a gram-negative bacterium.
 38. A compositionaccording to claim 33 wherein said MLA is extracted from a deep roughmutant strain of a gram-negative bacterium.
 39. A composition accordingto claim 36 wherein said 3D-MLA is extracted from a Salmonella bacteria.40. A composition according to claims 30 wherein said LPS is extractedfrom a Salmonella bacteria.
 41. A composition according to claims 33wherein said 3D-MLA is extracted from a Salmonella bacteria.
 42. Acomposition according to claim 34 wherein said bacterium is SalmonellaMinnesota.
 43. A composition according to claim 35 wherein saidbacterium is strain Salmonella Minnesota R595.
 44. A plurality ofcompositions of 3D-MLA having a consistent hexaacyl content.
 45. Aplurality of compositions according to claim 44 wherein said hexaacylcontent is consistently at least about 20 mol %.
 46. A plurality ofcompositions according to claim 44 wherein said hexaacyl content isconsistently between 20 mol % and 50 mol %.
 47. A plurality ofcompositions according to claim 44 wherein said hexaacyl content isconsistently at least about 50mol %.